The popularity of electronic cigarettes (E-cigarettes) is increasing worldwide. Marketing strategies have suggested that E-cigarettes can be used by tobacco smokers to help them quit, however, recent studies from 8 U.S. colleges indicate that 17% of E-cigarette users had never smoked a conventional cigarette. Each E-cigarette can provide 200-400 puffs, without interruption, which is equivalent to 2-3 packs of cigarettes. Nicotine, the main ingredient in E-cigarettes aerosol and at high concentration it is toxic and addictive. Given that E-cigarette aerosol may contain other harmful toxic compounds, regulatory agencies recommend caution in the use of E-cigarettes until their safety is better evaluated and documented. The initial interaction of E-cigarette aerosol mixtures occurs largely in the oral cavity, where nicotine and other compounds are expected to be most active and the exposure is most intense. Our preliminary data using commercially available E-cigarettes, demonstrate that the effects of E-cigarette aerosol on epithelial cells are profound. The effect of E-cigarette aerosol on the oral microbiome was significant as our 16S rRNA sequencing detected a shift in the salivary bacterial profile. Inflammatory mediators associated with periodontal health were higher in E-cigarette users than in nonsmokers. Furthermore, in vitro studies using a novel Electronic Cigarette Aerosol Generator (ECAG) and epithelial cells demonstrated that levels of transcripts that encode cytokines and chemokines were significantly higher in the group of cells which were first challenged by Porphyromonas gingivalis and then exposed to the E-cigarette aerosol than in cells exposed to either P. gingivalis only or P. gingivalis and air. These data suggest that E-cigarette aerosol can alter the homeostasis of the oral cavity through its direct effects on epigingival tissue and the oral microbiome. We hypothesize that E-cigarette aerosol mixtures disrupts the homeostasis of the oral microenvironment by altering the composition of the oral microbiome and increasing the expression of inflammatory mediators. We will conduct clinical and in vitro mechanistic studies in two integrated Specific Aims. In Specific Aim 1, we propose to conduct a prospective study in which we will enroll 120 age-matched subjects (40 in each of 3 groups: control nonsmokers, only tobacco smokers, and only E-cigarette users) to determine the effects of E-cigarettes aerosol on the oral microbiome and inflammatory mediators associated with periodontal disease. In Specific Aim 2, we will use a 3D epigingival tissue model with an ECAG to determine the impact of E-cigarette aerosols on epigingival tissue and inflammatory mediators when challenged by periodontal pathogens. We will apply novel clinical and in vitro approaches to reveal the oral health impact of E-cigarettes, which will address the research priorities of the NIH-NIDCR. This is the first comprehensive study designed specifically to identify microbiome and periodontal disease biomarkers for measuring and monitoring the adverse health effects of E-cigarette aerosol mixtures and for filling gaps in our understanding of the potential risks associated with E-cigarettes.